Longo, Ieda Maria

Pereira, Carlos Augusto

Ricci Júnior, Octavio

Silva, Ailton de Paiva e

English

Cadernos Espinosanos (E-Journal)

Rev. Inst. Med. top. São Paulo22(4) :180-183, julho-agosto, 1980uDc 593 t6t.t3AUTOMATED COMPLEMENT FIXATION TEST FOR THE DETECTION OFANTI.TRYPANOSOMA CRUZI ANTIBODIESCarlos Augusto PEREIRA (1), Ieda Marta If)NGO (2), Octavio AICCI JB, (2) end Ailto¡r de p¡iva, e SILVA (Z)SUMMARYAn automated method to detect TrypanosomÐ crozi antibodies is described,using complement fixation with an adequate automated equipment. Fifty five se-rum samples from patients lvhere Trypanosoma cruzi antiþodies were detectedby Almeida's technique for complement fixation and by fndirect Immunofluores-cence test, were studied by this method. The conclusion is that this automatedüechnique can be perfectly adapted to serology services in blood banks that aimto eliminate eventual diseases transmitted by blood donors.INTRODUCTIONSeeking the utilisation of automated equi-pments that have been designed for increa-sing rapidity and decreasing costs of the labo-ratorial tests, we made this work in order tostandardize a serological evaluation techniqueimportant in our environment.Several techniques are described and usedin the detection and the research of the contentof anti-Trypanosoma cruzi ¿¡fiþoflies3,l,s'0,r.These techniques present rather satisfactoryresults that are specific, sensitive and relati-vel¡r simple yet laking on rapidity which is veryimportant when handling a large number ofsamples, as is the case of the serology depart-ment of blood banks that aim to exclude even-tual diseases transmitted by blood donors.rffe standardized, in a Technicon automa-ted equipment, the automation in a complement fixation for detection of the anti-Îrypa.nosoma cruzi antibodies, according to a me-thod previously describedT. 'fn order to compare this technique withthe manual complement fixation test and indi-rect Ímmunofluorescence test, 55 patients thatshowed anti-Trypanosoma cruzi antibodies hadtheir serum samples analysed. We also ana-lysed serum samples from 10 normal indivi.duals.MATERIAL AND METHODS1) Sera - For the different reactions weutilised 55 serum samples of individuals thatshowed anti-Trypanosoma cruzi antibodies and10 sera samples of individuals from non ende-mic areas. The serum samples were collectedaseptically and kept under -20.C until the mo-ment of its use.2) Indirecú Immunofluotescence úest (IIT)- ft lvas performed according to the classictechnique of indirect immunofluorescence,described by CAMAF¿GOõ.3) Manual Complemenú Fixation Test(mCFT) - It was performed according to theAlmeida's technique t. 'We will not describe thetechnique since it is minutely reported in theoriginal work.4) Auúomaúed Complement Fixaúion Test(aCFT) - It was performed in the TechniconTmbalho tea'lizado no Departamento de Microbiologia e Imunologia da Faculdade de Ciências Médicas da S.Ðta Casade São Paulo. Rua Cesário Motta Jr., 112, 011ì21 São paulo, Bmsil(1) Professor de Microbiologia e Imunologia da Faculdade de Ciências Médicas da Santa Casa de São paulo(2) Estagiários da Faculdade de Ciências Médicas da Santa Casa d.e São paulo180PEREIRA, C, A.; LONGO, I. M.; RICCI JR., P. & SILVA, A. de P. e - Automated complement fixation test for the de-tection of anti-Trypanosom¿ cruzi antibodies. Rev. Insú. Med. trop. São P¿ulo 22:180"183, 1980.Autoanaþser systeme, designed for serologicalreactions, using a previously described techni-que z. fn brief, this automated test uses a con-tinous flow principle where all reagents are mi-xed automacfically and the results are showedon a recorder paper after a colorimeter pas-sage. The technique performed is correspon-dent to the recommended by the "LaboratoryBranch Complement Fixation" 2, which areshown in Fig. 1.COLOR]IYETER COLDRIMETERRECORDER5) Reagents - The reagents employed inthis reaction, such as methylic antigen, comple-ment, hemolysin, erythrocytes and total antigammaglobulin conjugate for immunofluores-cen€e, ïrere acquired from usual suppliers.RESULTSrüe performed the complement fixation test,in a total of 55 serum samples from patientswho showed anti-Trypanosoma cruzi antibodiesby the immunofluorescence and manual com-plement fixation tests, and in 10 serum sam-ples from normal individuals.The comparison between the results obtain-ed by these technique are presented in theTables I and II.Flow diågram öf the automated complement fixatlonm1lmlnU' UU JtrñO,42 AT,RO,60 SESAf"IPLERANTIGENCOflPLEMENTCOIYP LEIYENTHEI"]DLYTIC SYSTENHEITÓLYTIC SYSTEI{2,30The 10 serum samples, from normal indi-viduals, showed coherent results, that is, nega-tive by these two techniques studied.DISCUSSIONThe available techniques for the detectionof anti-Trypanosoma cruzi antibodies, althoughsensitive and precise, are not very rapid andtherefore not very suitable for blood banksserology laboratories which have high workload.'We believe that this automated equipmentfullfills the required needs for rapidity of exe-cution. We also believe that the standardiza-tion of this method, with commercially avai-lable reagents simplifies the execution and0,600, 60I AnF¡C. I181PEREIRA, C. A.; IONGO, I. M.; RICCI JR., P. & SILV.{, A. de P. e - Automated complement fixation test for the de-tection of anti-IlJ4lanosoma cruzi antibodies. Rev. Inst. Me¡I. trop. São Paulo 22:180-183, 1980'TABLE ICompa.rison between the results obtained by the acF'Îand mC!'T. in a total of 55 exarnined seraaCFT - Titersways. Besides the rapidity of execution (nearly40 tests per hour) of this automated comple-ment fixation method, it is possible to obtaina relative quantitative result; or, we can givethe quantitative result obtained vvithout pre-vious serum dilution, only with titers 2, 4, Band > 16 z, being an economicâl and easy wayto work with many samples.We believe that this method may be em-ployed for the detection of antibodies originatedby the other loacterial, parasitic or viral agents.This would greatly help the accomplishmentof a series of researches and evaluations thafare not being made actually due to the diffi-culty in performing the reaction in a largenumber of samples.RESUMOReação de fixagão do complemento automati'zada para detecgão de anticorpos anúi-Trypano-soma cruziUm método automa,tizado para a detecçãode anticorpos anti-Trypanosoma cruzi é des-crito. O método utiliza a técnica de fixaçãodo complemento em equipamento adequadopara a realizaçáo de testes automatizados. Fo-ram estudados por este método, 55 amostrasde soro de pacientes onde esses anticorpos fo-ram detectados pelas técnicas de complementodescrita por ALMEID¡ t, e teste de imunofluo-rescência indireta. Concluímos ser esta técni-ca automatizada perfeitamente adaptável a ser'viços de sorologia de Bancos de Sangue quevisam eliminar eventuais doenças transmitidaspor doadores de sangue.REFERENCESALI\4EIDA, J. O. - Reaçã.o de fixação de complementoem gotas sôþre placas pelo método das curvas iso-he-motÍticas. Eev. fnst. Med. tuop. São Paulo 5: 176-189,lvoó.ANON - Standardized diagnostic compleñrent fíxationmethod and adaptation to midotest. Puhlic HeslúhServ. Publ., 1965.ARAUJO, S. G. & BATISTA, S. M. - Observaçõessobre os testes de fixação de complemento e imuno-fluorescência indireta em doença de Chagas. Rev. Inst.Med. trop. São Paulo 11: 104-110, 1969.BIAGI, .F.; TAY, J. & MURRAY, R,. M. - La reacciónde fnmufluorescencia en el diagnóstico de Ia er¡fefm+dad de Chagas. Bol, Oflc. Panamer. 5?(2): 1964.2 48 16 TotalmC¡'TTiters(')1200160024003200480096001920042252I227/4 1/2(")1/16 U4r/8 7/4t/32 t/8L/16 U8u32 t/16t/64 7/32311ß3o5L2513I311930(') Evaluated in terms of serum necessary f.or SOE| of.lysis, a.r¡d determined by the value of the arigle ofinclination of immune complex-complernent regressionline.(") Dilutions oþtained with 3p and 6p of complement(cH50).TABTE IIComparison between the results obtained by the aCFT andIIT. in a total of 55 ex¿mined seraaCFT - Titers801603206401280o 2o213o63t4227I?gives better reproductibility of results amongdifferent laboratories that use the same me-thod.From the results we conclude that the IISis more sensitive than the other techniques,which is in accordance with current literature:,a'ó,8. fþs two different methods of complementfixation test have about the same sensitivity,although the titers are expressed in different1823.PEREIRA, C, ,q'.; LONGO, I. M.; RICCI JR., P. & SILVA, A. de P. e - á,utomated complement fixatlon test for the dstection of anti-Trypanosoma crud antibodies. Erv. Inst. Med. trop. São Paulo 22:180-18Í1, 1980,5. CAMARCIo, M. E. - Fluorescent antibody test for theserodiagnosis of American trjrpanosomiasis. TechnicaJmodificâtion employing preserved culture forms of Try-p&nosoma cruiz¡ irr a slide test. B.ev. Insú, Med. trop.São P¿ulo 8z 227-234,1966.CERISOLA, J. A.; ALVARES, M. & DE RISSIO, A.M. - Imunodiagnóstico da doença de Chagas. Fvoluçãosorológica de pacientes com doençÐ de Chagas. Rev.Inst. Med. trop. São Paulo t2: 403-411, 19?0.PEREIRA, C. A. 8z PEREIRA, O. A. C. - Pré-requisitopar¿ revacina4ão anti-rábica: Detecçã,o e avaliação daresposta imune humor&l à vâcinação passad.a, por pro-vas in viúro, BoI. Of. S¡nit, Panamer, 86 (3): i9?9.SADUN, E. H.; DU)<B{'RY, R. E. & WILLIAMS, J.S, - Fluorescent antibody test for the sero diagnosticof African and Americân trypanosomiasis in man. J.Paraslú. 49: 385-388, 1963.TECHNICON CORPORATION - New york, .farrytown,1976.Recebido para publicaqão em 14/12/1979.¡l67.183

Automated complement fixation test for the detection of anti-trypanosoma cruzi antibodies

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Automated complement fixation test for the detection of anti-trypanosoma cruzi antibodies

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